The present invention relates generally to solid phase methods for conducting specific binding assays upon sample fluids and more specifically to the use of chromatographic techniques in conducting such assays.
The use of specific binding assays has been found to be of great value in a variety of clinical applications. Various biological fluids and tissue samples can be analyzed for a wide variety of components such as drugs, hormones, enzymes, proteins, antibodies, DNA and RNA fragments and other biological material. Specific binding assays include those assays wherein an analyte is measured which is a member of a specific binding pair consisting of a ligand and a receptor. The ligand and the receptor are related in that the receptor specifically binds to the ligand, being capable of distinguishing the ligand from other sample constituents having similar characteristics. Immunological assays depend on reactions between immunoglobulins (antibodies) which are capable of binding with specific antigenic determinants of various compounds and materials (antigens). Specific binding assays may also involve DNA and RNA hybridization reactions wherein single strands of polynucleotides hybridize through hydrogen bond formation with strands of other polynucleotides comprising complementary sequences. Still other specific binding assays are known such as those involving hormone receptors which involve neither immunological reactions nor DNA hybridization.
Because the results of specific binding reactions are frequently not directly observable, various techniques have been devised for their indirect observation. Specific binding reactions may be observed by labelling of one of the members of the specific binding pair by well known techniques including radiolabelling and the use of chromophores, fluorophores and enzyme labels. Radiolabels, chromophores and fluorophores may be detected by use of radiation detectors, spectrophotometers or the naked eye. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system wherein a compound such as a dyestuff, is activated to produce a detectable signal.
Immunological assays are of three general types. In competitive binding assays, labelled reagents and unlabelled analyte compounds compete for binding sites on a binding material. After an incubation period, unbound materials are washed off and the amount of labelled reagent bound to the site is compared to reference amounts for a determination of the analyte concentration in the sample solution. A second type of immunological assay is known as a sandwich assay and generally involves contacting an analyte sample solution to a surface comprising a first binding material immunologically specific for that analyte. A second solution comprising a labelled binding material of the same type (antigen or antibody) as the first binding material is then added to the assay. The labelled binding material will bind to any analyte which is bound to the first binding material. The assay system is then subjected to a wash step to remove labelled binding material which failed to bind with the analyte and the amount of labelled material remaining is ordinarily proportional to the amount of bound analyte.
A third type of immunological assay technique involves agglutination reaction techniques and is exemplified by well-known assays for blood antigens and serum types. Immunological cross-reactivity between antibodies within serum and antigens presented on red blood cell surfaces is indicated by the formation of a three dimensional cross-linked network of red blood cells and antibodies. The agglutination of the serum/red blood cell mixture results in the formation of a pellet which can be visible to the naked eye.
These various assay procedures were originally performed according to liquid phase immunochemistry techniques wherein enzyme and radiolabelled reactions were carried out in liquid solution in apparatus such as microtiter plates. More recently, techniques and procedures have been adapted for carrying out "solid" phase assays wherein enzymatic and immunological reactions are carried out in solution on damp porous solid substrates.
U.S. Pat. No. 4,328,183 to Rosenfield, et al. discloses a solid phase blood typing procedure whereby a monolayer of lysed red blood cell ghosts is covalently bound to the walls of a plastic tube. The monolayer is then contacted with a serum sample and immunoadsorption of the antibodies present in the sample by the bound red blood cell ghosts occurs when the antibodies are reactive with antigens presented by the cell membranes. The antibody sensitized monolayer of blood cells can then bind a second layer of blood cells carrying complementary antigen in an agglutination reaction. If the immunological type of both the cell monolayer and the antibody layer are known, the formation or non-formation of a second cell layer can be used to indicate the immunological type of the cells forming the second layer. Conversely, if the immunological specificity of the first cell layer is known, the ability to form a second cell monolayer with the same cells can be relied on as a means for determining whether or not there had been formed an immunosorbed layer of antibodies specifically reactive with the antigens of the first cell layer.
U.S. Pat. No. 4,168,146 to Grubb, et al., discloses the use of test strips for carrying out assertedly "solid phase" sandwich-type immunoassays. The strips are formed of bibulous carrier materials to which antibodies have been attached by adsorption, absorption or covalent bonding. Preferred test strip materials include cellulose fiber-containing materials such as filter paper, ion exchange paper and chromatographic paper. Also disclosed are uses of materials such as cellulose thin-layer chromatography discs, cellulose acetate discs, starch and three dimensional cross-linked materials such as Sephadex (Pharmacia Fine Chemicals, Uppsala Sweden). Immunoassays are carried out by wetting the test strips with measured amounts of an aqueous solution containing the suspected antigen. Antigen molecules within the test solution migrate by capillary action throughout the test strip, but because the bound antibodies retard the migration of the antigens for which they are specific, the extent of migration of the antigen molecules over a fixed time period is related as a function of the antigen concentration in the test solution. The antigen-containing areas of the diagnostic device are then indicated by the addition of labelled antibodies.
U.S. Pat. No. 4,517,288 to Giegel, et al. discloses methods for conducting solid phase immunoassays on inert porous materials. The patent discloses immunologically immobilizing a binding material within a specified zone of the porous material and applying the analyte to the zone containing the immobilized binding material. A labelled indicator material which will bind with the analyte is then applied to the zone where it will become immobilized in an amount correlated to the amount of analyte in the zone. A solvent is then applied to the center of the zone to chromatographically remove the unbound labelled indicator from the zone so that the amount of labelled indicator remaining in the zone may then be measured.
Deutsch, et al., U.S. Pat. No. 4,361,537 discloses test devices for the performance of specific binding assays such as radiolabelled competitive binding assays comprising a strip capable of transporting a developing liquid by capillarity which has a first zone for receiving a sample, a second zone impregnated with a first reagent capable of being transported by the developing liquid and a third zone impregnated with a third reagent. In addition, the devices comprise a measuring zone and a retarding element which may be either the second reagent or the material of the strip. The first reagent is capable of reacting with one of the group consisting of (1) the sample, (2) the sample and the second reagent, or (3) the second reagent in competition with the sample, to form a product in an amount dependent on the characteristic being determined. A sample is contacted with the first zone and the strip is then dipped into the developing liquid to bring about transport of the sample and the first reagent to form the reaction product. The retarding element slows transport of either the product or the first reagent (the moving reagent) to spacially separate the two and the amount of the moving element is then measured at the measurement location.
U.S. Pat. No. 4,435,504 to Zuk, et al. discloses a chromatographic immunoassay wherein the distance at which a border is formed from one end of the chromatograph is indicative of the quantity of analyte present in a sample. The analyte which is a member of a specific binding pair is immunochromatographed on a bibulous carrier to which its binding partner is nondiffusively bound and a variety of protocols are utilized to provide for delineation between the region to which the analyte is bound and the region free of analyte. According to one protocol, the analyte is chromatographed in the presence or absence of a labelled binding conjugate where the label is a member of an enzymatic signal producing system which includes one or more enzymes. If the labelled conjugate is not chromatographed with the analyte, the conjugate is applied to the chromatograph where it will bind to the chromatograph in proportion to the amount of analyte present. Similarly, if the labelled conjugate is chromatographed with the analyte, then the conjugate will bind to the analyte in proportion to the amount of analyte present at that position. The labelled conjugate can be an enzyme member of a signal producing system which can include chromophores, phosphors, fluorescers and chemiluminescers as well as coupled enzymatic signal systems. Where a coupled enzyme system is utilized, a second enzyme capable of reacting with the product of the first enzyme catalyzed reaction to form a detectable product may be chromatographed with the analyte solution or may be added to the test strip after chromatography of the analyte.
European Patent Application No. 164,194 (published Dec. 11, 1985) discloses improvements on the methods of Zuk, et al. in that transported chromatographic materials have substantially the same rate of traversal along the longitudinal edge of the chromatographic strip as along the body of the strip. This allows the chromatographic transport front to remain substantially flat rather than concave.
Of interest to the present patent application are two published patent applications of the inventor. U.S. Pat. No. 4,452,901 to Gordon discloses the use of porous nitrocellulose supports for immobilization of proteins. It is disclosed that such nitrocellulose sheets may be utilized in immunoassay procedures if the residual binding capacities of the nitrocellulose sheets are saturated by blocking treatment with one or more types of proteins, different from those immobilized and not cross-reactive with any of the antibodies subsequently used in the assay.
Of further interest to the background of the invention are the disclosures of Gordon, EPO Application 63,810, published Nov. 3, 1982, relating to devices for conducting immunological assays. The devices consist of a porous solid support containing a preselected array of delimited adsorption areas of antigens, antibodies or both, wherein residual adsorption sites on the substrate are saturated by protein blocking agents such as bovine serum albumin which do not cross-react with the antigens or antibodies employed in the assay. The porous supports are disclosed to have sufficient surface porosity to allow access by antibodies and surface affinity suitable for binding antigens. Such supports are disclosed to be selectable from a variety of natural and synthetic polymers and derivatives but are preferably nitrocellulose sheets 0.1 mm thick with pore size between about 0.15 .mu.m and about 15 .mu.m. Antigens or antibodies are applied to the porous solid support by direct contact followed by incubation with blocking agents. Assays for detection of unknown antigens or antibodies are then carried out through use of labelled antibodies which may also be anti-immunoglobulin antibodies. Results of single or multiple assays are determined by detection of the labelled antibodies.
Various specific binding assay techniques are also well known for the detection of specific DNA and RNA sequences. Such assays utilize nucleic acid hybridization procedures wherein complementary polynucleotide sequences of single stranded nucleic acid polymers recognize each other and interact to form a stable duplex structure. Southern, J. Mol. Biol. 98, 503-517 (1975) discloses procedures wherein DNA molecules separated by gel electrophoresis may be transferred from agarose electrophoresis gels to nitrocellulose filter paper. The DNA fragments may then be hybridized to radiolabelled RNA fragments for detection of particular sequences.
Falkow, et al., U.S. Pat. No. 4,358,535 discloses methods useful for the detection of DNA sequences associated with the infectious microorganism is isolated and fixed in a single stranded denatured form to an inert support such as nitrocellulose. A labelled polynucleotide probe specific for a DNA sequence characteristic of a pathogenic product suspected of being present in the clinical sample is contacted with the sample DNA under hybridizing conditions. The support is then washed to remove any unhybridized probe material and the presence of any remaining hybridized probe material is indicative of the presence of pathogen.
Dunn, et al., Cell, 12, 23-36 (1977) discloses an alternative hybridization procedure known as sandwich hybridization. According to this procedure, sample RNA is hybridized to defined DNA fragments which are bound to nitrocellulose filter paper supports such that the 3' or 5' end of the RNA protrudes as a single-stranded tail. DNA sequences complementary to the "tail" sequences can then be determined by treatment with specific fragments of labelled DNA under hybridizing conditions.
In addition to the various specific binding assay procedures known in the prior art, there also are known numerous assay procedures involving the diffusive or chromatographic transport of assay reagents. Forgione, U.S. Pat. No. 3,875,014 discloses solid phase test indicators for the determination of concentrations of the enzyme aspartate aminotransferase (AST) in sera utilizing a pair of reactions. In the first reaction AST catalyzes the reaction of L-aspartic acid and alpha ketoglutaratic acid to form oxaloacetate. In the second reaction, oxaloacetate reacts with a diazonium salt to form a colored reaction product. The test indicator of Forgione, comprises a pair of bibulous materials, adhered to each other with an adhesive which is selectively permeable to oxaloacetic acid. The first material is impregnated with the substrates L-aspartic acid and alphaketoglutaric acid. The second material is impregnated with a dried diazonium salt dyestuff. The device is contacted with sera which, if it contains AST, catalyzes the reaction of the substrates to form oxaloacetic acid. Oxaloacetic acid then diffuses through the adhesive barrier to the second strip and activates a color reaction with the diazonium salt which may be compared against standards.
Campbell, U.S. Pat. No. 3,893,808 discloses a test strip for the detection of lead contamination in unleaded motor fuels. The test strip comprises a paper strip having three zones. The first zone is impregnated with iodine, while the second zone is treated with a mixture of iodine and potassium iodide. A sample of motor fuel to be tested is applied to the strip and is transported by means of capillary action through the first and second zones to the third zone to which a dithizone indicator solution is then added. Any organic lead present in the motor fuel is converted to inorganic lead iodide on the surface of the strip and this is detected by reaction with the dithizone indicator to form a lead dithizonate complex with a characteristic color.
Alberty, et al., U.S. Pat. No. 3,895,914 discloses a test strip for the detection of barbituric acid and barbituric acid derivatives in a biological fluid. The strip comprises a bibulous paper strip having three zones. The first zone is impregnated with acid in order to acidify sample fluids applied thereto. The second zone is impregnated with alkaline buffered mercuric acetate capable of reacting to form a barbituratemercury complex. The third zone is impregnated with a mercury indicating compound such as diphenyl carbazone. A sample of fluid to be tested is applied to the first zone and the strip is dipped in solvent. Barbiturates present in the sample will react to form a barbiturate-mercury complex which will be transported to the third zone and will react with the mercury indicating compound.
Kallies, U.S. Pat. No. 4,298,688, discloses an assay device for the determination of glucose levels in biological fluids. The device comprises a paper test strip demarcated into a measuring zone which may be untreated, a reaction zone containing glucose oxidase, and a detection zone containing peroxidase and indicator substances such as o-tolidine and Orasol yellow. The material to be assayed is allowed to diffuse through the measuring zone to the reaction zone, wherein any glucose will react with the glucose oxidase, and then to the detection zone wherein a color reaction will take place, the degree of which depends on the extent of reaction carried out in the reaction zone. Water may be used to assist the diffusion of the test materials through the device and the test strip may also be enclosed within a glass capillary tube.
Fogt, et al., U.S. Pat. No. 4,444,193 discloses a quantitative test device for the measurement of chloride levels in sweat. The device, which is designed for use in screening for cystic fibrosis comprises a flat patch which when placed on the skin of a subject collects a fixed amount of sweat. The patch consists of two concentric circular reaction areas of chemically treated absorbant material. The sweat sample is introduced at the center of the inner circular reaction area which contains a chemical composition such as silver phosphate capable of reacting with all chloride in the sweat sample below a predetermined concentration in order to "screen out" a threshold quantity of chloride. The outer ring-shaped reaction area contains a chemical composition such as silver chromate which is brown in color and which reacts with any chloride reaching it to form white colored silver chloride and produce a color signal indicating the presence of chloride in excess of the predetermined threshold.
Of interest to the present invention is the disclosure of Wieland and Determann, J. Chromatog., 28, 2-11 (1967) relating to the use of Sephadex gels employed in a thin-layer chromatography format for separations of proteins. While Sephadex is not known as a conventional thin layer chromatography substrate and conventional thin layer chromatography is not practiced for the separation of proteins, highly cross-linked particles of Sephadex G-25 were used in ascending thin layer chromatography formats but a change in manufacture of Sephadex to bead form made ascending chromatography unworkable as the particles would not adhere and cohere satisfactorily. The use of large pore Sephadex types G-100 and G-200 in descending thin layer chromatography formats to the separation of proteins is also disclosed.
Morris, J. Chromatog., 16, 167-175 (1964) also discloses use of Sephadex type G-100 and G-200 plates in descending chromatography for proteins. The disclosure notes such transport is slow, however, stating that under "optimal operating conditions", human CO-haemoglobin should migrate only about 70 mm in approximately 4 to 5 hours.
Despite the great advances that have been made with respect to specific binding assay techniques in recent years, there still remain significant opportunities for improvement of these techniques. A particular limitation of current assay techniques is the requirement of numerous addition and wash steps. These steps, required to prevent undesired cross-reactions and remove excess reagents and interfering substances, complicate the procedure and effectively limit the type and level sophistication of analytical procedures that may be carried out. Elimination or reduction of the number of washing and addition steps which must be carried out by technical personnel will not only reduce time and expense of conducting assays and analyzing assay results, but will also reduce the difficulty of automating result analysis. For these reasons, new systems involving solid phase assay devices requiring a minimum number of addition and washing steps are highly desired. Such devices would preferably be susceptible to use in conducting assays for a wide variety of materials and would be capable of providing for the performance of a complex sequence of reactions in an essentially automatic manner.